RAT-LAMP: Design and Characterisation of Conchimeric Primer Architectures Toward High-Density Solid-Phase Loop-Mediated Isothermal Amplification

Solid-phase loop-mediated isothermal amplification (LAMP) offers a pathway toward low-cost, in-tegrated molecular diagnostics, but practical implementation is limited by poor primer bioavailabilityand insufficient surface primer payload. We present RAT-LAMP (RNaseActivated Trigger for Loop-Mediated isothermal AMPlification) and a conchimeric primer architecture as a dual strategy to over-come these constraints. Conchimeras are concatenated multi-primer oligonucleotides composed of distinctLAMP primer species separated by RNA spacer residues, enabling on-demand primer release throughRNase-mediated cleavage during amplification. The adoption of conchimeras reduces the number ofdistinct LAMP primer species competing for a fixed surface area from six to three.Chimeric and 81 to 84-base conchimeric primers were designed and evaluated targeting syntheticDengue virus 1 DNA. The functionality of the designed primers was demonstrated for simultaneousRNase and polymerase activity under standard LAMP conditions. RAT-LAMP restored chimeric primerperformance to near-unmodified levels (time-to-positive: 2.81 vs. 2.59 minutes for unmodified primers),confirming the feasibility of on-demand primer release. Conchimeric primer amplification was feasiblefollowing spacer optimisation (rCrG and rUrG designs), with RNase supplementation reducing batch-to-batch coefficient of variation by ∼ 4-fold (16.47% to 3.76%). Amplification using shorter conchimeras con-taining FIP and BIP confirmed that the rate-limiting primers (FIP and BIP) should not be concatenatedwith other primers. Notably, primers modified via 5′-phosphorylation for EDC surface immobilisationproduced a statistically significant improvement in amplification speed over unmodified primers (TTP:2.36 minutes; p-value =0.0006). Together, RAT-LAMP and conchimeric primer architectures establishthe component feasibility of high-density primer immobilization for solid-phase LAMP. They simultane-ously address primer availability and surface loading constraints without increasing workflow complexity.Adoption of a three-species conchimeric system increases primer cost by only 1.4-fold over unmodifiedprimers (£0.010/ test - conchimera vs £0.007/ test - unmodified). Regardless of the cost increase, at£0.021/ test, the adoption of conchimeras for solid-phase primer delivery would not significantly impactconsumable cost